A normal functioning human nervous system requires the interconnection of billions of neurons. Improper formation or maintenance of these connections leads to neurological abnormalities that result in a number of mental diseases and disorders. How are these circuits assembled and integrated? The semaphorins are one of the largest protein families involved in the formation and maintenance of axonal connections. Semaphorins are phylogenetically conserved secreted and transmembrane proteins found in invertebrates and in vertebrates. Semaphorins utilize plexins, a family of large transmembrane proteins found on the axonal surface, as receptors to direct their effects. How plexins actually transduce semaphorin signals is poorly understood but is of importance for learning how semaphorins sculpt and maintain the nervous system. So what strategies will further define these important mechanisms by which semaphorins and plexins direct neural connectivity? Work over the past twenty years has revealed that the molecular mechanisms of axon guidance and connectivity are remarkably well-conserved between simple and complex animals. Simple animals like flies use many of the same axon guidance signals as mammals. In light of this conservation, the goal of my research program is to focus on a small group of axons within the simple nervous system of the fly embryo and characterize the molecules and mechanisms that guide them to their targets. Using this strategy, I recently identified a new family of intracellular proteins, the MICALs, that are critical for directing semaphorin/plexindependent neural connectivity. There is one MICAL gene in simple organisms like flies, while three separate MICAL genes are found in mammals including humans that are also important for mediating the effects of semaphorins and plexins. Interestingly, MICAL proteins contain several regions known to interact with the cytoskeletal machinery necessary for allowing axons to grow, navigate, and form their connections. MICALs also contain an oxidoreductase domain, the integrity of which is required for Semaphorin axonal connectivity. The presence of this oxidoreductase domain implicates for the first time oxidation-reduction signaling mechanisms in semaphorin-mediated connectivity. One important question that is the focus of this proposal is to identify the molecules through which MICAL steers an axon. Initial insight into this question has come with our recent identification that MICAL interacts with the SH3-domain containing protein Cas in neurons. Cas is a critical regulator of actin cytoskeletal dynamics in non-neuronal cells and we find that Cas and MICAL link Plexins and integrins to mediate axon guidance. Our preliminary results now reveal that Cas interacts with a specific mediator of G protein signaling suggesting the possibility that MICAL and Cas play a role in regulating GTPases in navigating axons. We will use in vivo genetic and biochemical approaches and the model fly axon system to test the hypothesis that specific GTPases and their regulators are mediators of axon navigation and play an important role in the intracellular signaling mechanisms utilized by semaphorins during axon guidance.